Several lines of experimental evidence, including our own work, indicate that disruption of certain aspects of intracellular cholesterol homeostasis in various cell types (e.g. macrophage, -cell, adipoctyes) can lead to pathological processes preceding Type 2 diabetes mellitus (T2DM). Our recent transcriptomic study of human monocytes (N=1,264) corroborates these findings, and this study specifically identified a co-expressed cholesterol metabolism network (CMN) of genes whose alterations were associated with T2DM (ptrend: 5.07x10- 10). This network contained 11 functionally coupled genes related to cholesterol metabolism, with up-regulation of cholesterol uptake and synthesis, and down-regulation of cholesterol efflux a molecular profile expected to increase intracellular cholesterol. Furthermore, our data along with in vitro and in vivo data suggest that the effects of two important risk factors for T2DM - obesity and inflammation, may be mediated through alterations in this network. In addition, we identified several DNA methylation sites that potentially regulate expression of the CMN genes and were associated with T2DM. However, the strength of this inference linking the molecular features of CMN toT2DM risk is limited by the cross-sectional nature of our human data. Here our goal is to elucidate the temporal relationship between molecular features of the CMN and T2DM onset and to further investigate how the predicted functional consequences of the altered network in monocytes relate to the development of T2DM. We hypothesize that the molecular features of the CMN related to up-regulation of cholesterol uptake and synthesis and down-regulation of cholesterol efflux and increase in intracellular cholesterol content will predict a higher risk for developing future T2DM If confirmed, results will provide a rationale for developing a systematic approach to modulate this CMN, rather than individual genes, for optimizing the prevention and treatment of T2DM. Based on these preliminary data, and taking advantage of the well-phenotyped Multi-Ethnic Study of Atherosclerosis (MESA) cohort with existing genomic, DNA methylomic, and transcriptomic data on 1,264 CD14+ (primarily monocyte) samples, we now propose to carry out analyses of additional monocyte samples (N=1,536), a follow-up assessment of T2D status, and in vitro characterization of monocyte functions to achieve the following specific aims: 1) To independently replicate cross-sectional associations of T2DM with the molecular features of CMN (DNA methylation and mRNA) in monocyte samples; 2) To determine whether the molecular features of CMN (DNA methylation and mRNA) can predict incident T2DM in a 7-year follow-up; and 3) To determine whether differences in intracellular metabolism which would be predicted from the CMN alterations can predict incident T2DM. The integration of genetic, epigenetic, transcriptional, and clinical data along with in vitro experimental studies may provide novel mechanistic insights concerning the role of cellular cholesterol metabolism in susceptibility to T2DM, possibly leading to new prevention or treatment strategies.